Structural proteins in the egg envelopes of dragonflies, Sympetrum infuscatum and S. frequens

Insect Biochem., 1974, Vol. 4, pp. 99 to 1 II. Pergamon Press. Printed in Great Britain

99

STRUCTURAL PROTEINS IN THE EGG ENVELOPES OF DRAGONFLIES, SYMPETRUM INFUSCATUM AND S. FREQUENS HIROYA KAWASAKI, HITOSHI SATO

AND

MOTOKO SUZUKI

Department of Biochemistry, School of Dentistry, Iwate Medical College, Morioka

020,

Japan

(Received 17 April 1973; revised 25 May 1973) ABSTRACT The egg envelopes of the dragonfly, Sympetrum infuscatum, consisted of three layers, the vitelline membrane (the innermost layer) and the inner and outer layers of chorion, their thickness being about 9, 0'3 and 3 fLm. respectively. The major constituents of the envelopes were structural proteins, and they were divided into three different protein fractions. The two fractions which originated from the chorion were soluble in a thiol-urea solvent, and they were obtained as soluble S-carboxymethyl derivatives, CM-fractions 1 and II. Both of them contained a large amount of Scarboxymethylcysteine, but they differed from each other remarkably in the contents of amide, glycine, alanine and sugars. The average molecular weights of CM-fractions 1 and II were estimated to be 8900 and 15 000 respectively. The constituent of the vitelline membrane was insoluble in the thiol-urea solvent and was referred to as the' insoluble residue'. This protein fraction did not contain cystine at all but contained dityrosyl and trityrosyl residues (4 fLmoles and 1 fLmole per 100 mg. of sample respectively) as another type of intermolecular cross-link. The approximate proportions of CM-fractions l, II and insoluble residue in the egg envelopes in the native state were assumed to be 1 : 3 : 14· The yellow colour of the newly laid eggs changed to dark brown after exposure to air. This darkening pro cess was found to occur in the vitelline membrane. The insoluble residue obtained from the dark eggs was also dark brown in colour and was more resistant against the hydrolysis by acid, alkali and proteolytic enzyme than was the yellow insoluble residue. The contents of tyrosine, lysine and histidine in the dark insoluble residue were markedly lower than those in the yellow residue, without any appreciable difference in the contents of other amino-acids. Similar changes were observable, though to a lesser extent, when the yellow insoluble residue was incubated with a mushroom tyrosinase. Thus, the chemical structure of the vitelline membrane, which had been stabilized by dityrosyl and trityrosyl residues, was assumed to be further reinforced during the darkening pro cess by the mechanism of ' self-tanning' . The results obtained from the eggs of another species of dragonfly, S. frequens, generally agreed well with those described for S. infuscatum material, with one exception that the CM-fraction II of S. frequens was obtained as two subfractions, CM-fractions lIa and lIb. OUR previous work on the egg-shells or chorions of 8 species of silkworm (2 from the family Bomhycidae and 6 from Saturniidae) (Kawasaki, Sato & Suzuki, 197Ia, 1972) and of the oriental garden cricket, Gryllus mitratus (Kawasaki et al., 1971h) indicated that these shells were mainly made of 2 or 3 groups of different structural proteins. The major structural proteins in the shells had a chemical structure stahilized hy the intermolecular disulphide linkage, and they could he dissolved in a thiol-urea solvent. The

100

KAWASAKI AND OTHERS

Inseet Bioehem.

chemical and physical properties of the major protein fractions were similar in aIl species of silkworm studied, including similar contents of cysteine (half cystine) (5.86'9 moles per cent of the total amino-acids), but the egg-shells of the 2 species of Bombycidae contained an additional protein fraction which was unique in its high content of cysteine (29'4-30·6 moles per cent). The major protein fraction of G. mitratus egg-shell also contained cysteine (1·8 moles per cent), but unlike the silkworm major proteins it was a phosphoprotein with high content of serine (35'7 moles per cent) and phosphorus (nearly equimolar to serine). These shells also contained a fraction of thiol-urea-insoluble proteins as the minor constituent (1-17 per cent of the total amount of the structural proteins), and its chemical composition differed even among the silkworms. These findings had led us to a comparative study on the insect egg-shells to look for more unusual structural proteins and to obtain basic data on the relationship between these proteins and the different protective functions of the shells. As part of such a study this paper deals with the egg envelopes (the vitelline membrane and chorion) of 2 species of dragonfly, Sympetrum infuseatum Selys and S. frequens Selys. MATERIALS AND METHODS MATERIALS

Dragonflies of the z species, S. infuscatum and S. frequens, were caught in Morioka in August and September. Most of the females began oviposition soon after being caught and held by the wings, and finished it in several minutes. The eggs, yellow in colour, were collected in a test-tube that was filled with water, freed from contamination, air-dried on filter-paper for about 1 hour, weighed, and stored frozen until use. The yellow colour did not change while kept in the frozen state ( - zoo C) even for several years. However, when the eggs were collected without water or the eggs collected in water were air-dried for longer than a few hours, the colour gradually changed to brown and finally became dark brown after Z4 hours of exposure to air. Such dark brown eggs were also used in the present study, and these will be referred to as the' dark eggs'. Without this reference the data are those obtained from the yellow eggs. PRELIMINARY FRACTIONATION OF THE EGG

Water-soluble and 0'01 M NaOH-soluble substances were removed from the egg by a preliminary fractionation as described previously for the silkworm eggs (Kawasaki et al., 1971a). The amount of proteins extracted from the crushed eggs with chilled water was 26'2 per cent of the dry weight of the egg in S. infuscatum and ZS'z per cent in S.frequens. The yield of proteins which were then removed from the egg with chilled 0'01 M NaOH was 0'5 and 1'5 per cent in S. infuscatum and S.frequens respectively. The residue of the extractions was washed with water, ethanol and ether before drying in vacuo over P 205' This fraction, designated the' scleroprotein fraction', represented 6'7 per cent of the dry weight of egg in S. infuscatum and 5'0 per cent in S. frequens. DISSOLUTION OF THE SCLEROPROTEIN FRACTION

As described for the silkworm materials (Kawasaki et al., 1971a), the scleroprotein fraction was extracted with an effective thiourea solvent, and the dissolved proteins were converted to S-carboxymethyl derivatives. The scleroprotein fraction (761 mg.) of S. infuscatum was stirred at 30° C. for 1 hour with zo ml. of a solvent consisting of urea (8 M), dithiothreitol (o'oz M), ethylenediaminetetra-acetate (5'7 mM) and Tris buffer (o·z M, pH 8'6). After centrifugation at 8700 g for 10 minutes, the precipitate was extracted three times more with s-ml. portions of the solvent. The insoluble material was washed with water, ethanol and ether, and dried in vacuo over P aü 5 • This protein fraction, slightly yellow in colour, was referred to as the' insoluble residue'. Its yield was 568 mg.

1974,4

101

PROTEINS IN DRAGONFLY EGG ENVELOPES

The extracts were combined and subjected to S-carboxymethylation by the use of sodium mono-iodoacetate as described previously. The solution was dialysed against water and finally freeze-dried to give 175 mg. of white powder. This was designated 'CM-fraction'. Similarly, from 301 mg. of the scleroprotein fraction of S. frequens 227 mg. of the insoluble residue and 71 mg. of the CM-fraction were obtained. The insoluble residue obtained from the dark eggs was also dark brown in colour, whereas the CM-fraction was white. The relative yields of the insoluble residue and CM-fraction were similar to those of the yellow eggs, although the yields of these fractions were higher than those from the yellow eggs by about 10 per cent, probably owing to the dehydration of eggs during the darkening process in air.

0.02

A 0.5 .... .... .. , .... 0.4 '

:::::L0.01

E

L()

1"N

,,

-6

0.3 U

,, ,,

0

0.2

+-

Il

0

Z

'0 c 0

Cl)

u

8

C 0

-e0 0.01

,

. . " ...... ,,.

E

0.5 0.4 0.3


'Q ê: Cl) u

ES

U

0.2

o

50

FractÎal no. FIG. I.-Column chromatography on DEAE-cellulose of CM-fraction of S. infuscatum (A) and of S.frequens (B). The CM-fraction of S. infuscatum (40 mg.) or that of S.frequens (35 mg.) was applied to the column (0'9 x 6'5 cm.) of DEAE-cellulose (Servaj capacity, 0'74 mequiv. per g.) buffered with 0'2 M NaCljlo mM citrate buffer, pH 5. A gradient elution was obtained by using 500 ml. of the above-mentioned buffer in the mixing chamber and 0·8 M NaCl/lo mM citrate buffer, pH 5, in the reservoir. Elution was carried out at 20° C with an approximate flow rate of 0'3 ml. per minute, and 3-ml. fractions were measured for ultraviolet absorption. - - - , Concentration of NaCI. FRACTIONATION OF THE CM-FRACTION

Since free-boundary electrophoresis showed that the CM-fraction of S. infuscatum consisted of two electrophoretically separable components, it was fractionated into two on a column of DEAEcellulose. A typical result is illustrated in Fig. lA. The minor protein fraction, which was eluted first from the column, was referred to as 'CM-fraction l'and the major fraction, eluted later, as

102

KAWASAKI AND OTHERS

Inseet Bioehem.

'CM-fraction II'. CM-fractions 1 and II were dialysed against water and freeze-dried. The ratio of yields of CM-fraction 1 to II was about 1 : 3 in both of S. infuscatum and S.frequens. However, as shown in Fig. lB, CM-fraction II of S.frequens was eluted as two subfractions, lIa and lIb, in an approximate proportion of 1 : 2. PHYSICAL METHODS

Free-boundary electrophoresis, ultracentrifugal analyses and determination of the partial specific volume were carried out as for silkworm materials (Kawasaki et al., 1971a). Acrylamide gel electrophoresis was carried out by the method described by Davis (1964), using 7'5 per cent gel. Owing to the poor content of basic amino-acids in the present samples, CMfractions 1 and II, standard staining methods with acid dyes did not give satisfactory results. Therefore, the gels were placed in alper cent solution of toluidine blue for 15 minutes without previous fixation of protein bands, washed with water repeatedly, and photographed. ANALYTICAL METHODS

An automatic amino-acid analyser, Hitachi KLA-3B, was used for the assay of amino-acids, amide and hexosamines. Samples were hydrolysed in an evacuated tube with 6 M HCI at 100 C for 6, 24 and 72 hours. The 6-hour hydrolysates were used for the assay of amide and hexosamines and the others for amino-acids. Since the values for serine and threonine found in the 24- and 72-hour hydrolysates did not show a considerable difference, the values for 24-hour hydrolysis were shown without correction for destruction during hydrolysis. Tryptophan content of the scleroprotein fraction and insoluble residue was determined by the method of Matsubara & Sasaki (1969), and that of the CM-fractions by the method of Goodwin & Morton (1946). Detection and assay of neutral sugars in the protein fractions were carried out as described by Masamune & Kawasaki (1956). Total nitrogen, phosphorus, moisture and ash were assayed as described previously (Kawasaki et al., 1971a). 0

IDENTIFICATION AND QUANTITATION OF DI- AND TRITYROS1NE IN PROTEIN FRACTIONS

Standard di- and trityrosine were prepared according to Gross & Sizer (1959), incubating Ltyrosine with hydrogen peroxide and horse-radish peroxidase. Acidified reaction mixture was concentrated in vacuo to a small volume and was subjected to ascending paper chromatography on Toyo filter-paper No. 50 with the solvent mixture, n-butanol/acetic acid/water (4 : 1 : 2, by volume) at room temperature for about 20 hours. Dityrosine (RF 0'26) and trityrosine (RF 0'17) were located by their blue fluorescence under ultra-violet light and were eluted from the filterpaper with water. They were further purified on a column of DEAE-cellulose as described by Andersen (1966). Protein fractions were hydrolysed in an evacuated tube at 100 0 C with 6 M HCI for 24 hours. After removal of HCI by repeated evaporation in vacuo, the hydrolysates were subjected to the same procedure described above to purify the standard compounds. The paper chromatograms of the hydrolysates from CM-fractions 1 and II did not show any blue fluorescent area, but the hydrolysates from the insoluble residues contained the two fluorescent compounds which migrated identically with standard di- and trityrosine on the filter-paper. Elution patterns of these compounds from the DEAE-cellulose column were also similar to those of the standards. The identity of the two fluorescent compounds was confirmed by cochromatography with the standard compounds on columns of DEAE-cellulose and cellulose phosphate (Andersen, 1970); on filterpaper with four different solvent mixtures (Andersen, 1966); and by their characteristic ultraviolet absorption spectra in acid and alkaline conditions (Andersen, 1966). Quantitation of di- and trityrosine in the protein hydrolysates was carried out also with the preliminary purification by paper chromatography and the chromatography on a DEAE-cellulose column. After the column chromatography, the fractions of di- or trityrosine were combined, concentrated when necessary, and acidified to pH 2 with HCL The extinction at their maximum absorption was measured, and the concentration was calculated from the molar absorption coefficients reported by Andersen (1966). Approximately 25 mg. of proteins were used for aech assay. Comparable amounts of bovine serum albumin, added with known amounts of di- and trityrosine, were subjected to the procedure to correct for the loss during this process, and the average recovery was found to be about 85 per cent.

1974,4

PROTEINS IN DRAGONFLy EGG ENVELOPES

1°3

RESULTS NATURE AND CHEMICAL COMPOSITION OF THE SCLEROPROTEIN FRACTION

According to the electron microscopie studies of Beams & Kessel (1969) on the ovarian egg of Aeschner species and by Matsuzaki (1971) on that of S, frequens, the oocyte of the Table r.-CHEMICAL COMPOSITION OF THE SCLEROPROTEIN FRACTIONS AND OF THE PROTEIN FRACTIONS OBTAINED FROM THE SCLEROPROTEIN FRACTIONS S, infuscatum ......

...... ......

.:: ,8 ... u

.:: ,8 ...

J::1

J::1

Cf.l~

U

U

Glucosamine Galactosamine

Trace

1 1

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine ! Cystine* Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

41 52 4 20

,S ....0 Cl)

CONSTITUENTS

...0..0.:: ...Cl) ...U o'~

......

«l

u ...

Dityrosine Trityrosine Amide Ash (per cent) Total nitrogen (per cent) Nitrogen recovered (per cent)

II

23 31 IlO 259 128 45 10 13 8 16 83 3 1

«l

:;s

u

«l

:;s 3 5

S,frequens ~

,S

,t:J

..a0

Cl)

::0 ~

'"

.::

....0 Q)

Cl)

...... :3

..a"1:l ~~ «l Cl) .:: Cl) ......'" ~'" O~ o'~

Trace Trace 0 0

58 3 30 0 0 71 55 6 6 2 3 18 22 17 41 6 6 23 33 II 80 10 56 100 II 30 31 120 122 60 72 152 25 1 340 245 160 15 15 8 73 180 218 0 0 8 10 21t 9 1 6 18 17 14t 13t 5 5 6 46 39 7 31 34 103 54 0 0 3 3 0 5 9 Trace

.::

...

......

......«l ......

......

.::0

.:: ,8 ... u

.:: ,8 ...

J::1

J::1

U

U

oB

«l

0..0 ...... u

J::1

Cf.l~

U

O'~

Cl) ...... U

«l ...

:;s

«l

:;s

,t:J

Cl)

t-1

::0

..a0

Cl)

::0 ~ '".::

u

«l

Cl)

..a"1:l ..... :3 ~~ «l Cl) $:l Cl) ......'" ~'" O~

:;s

o'~

1 2

Trace Trace Trace Trace 2 19

0 0

43 51 4 21 15 27 33 109 264 124 42 12 13 13 16 85 3 1

Trace Trace Trace 0 0 0 10 7 5 6 32 44 25 49 41 84 23 55 100 16 4 56 78 74 134 390 3 19 22 23 74 15 1 21 3 186 10 st 9 0 1 5 12t 43t 47t 29 41 41 39 30 30 0 0 0 6 6 5

57 66 3 18 6 17 32 Il9 259 15 6 0

29 54 3 17 6 17 34 Il4 256 161 0

II

II

16 6 8 101 3 Trace

18 6 7 47 3 0

Trace Trace

3 1

0 0

0 0

4 1

3

3 1

0 0

0 0

0 0

3 1

3 1

21

121

19

16

18

23

102

23

15

19

23

6'2

8,6

4'6

2'4

0'2

3'2

4'9

5'4

1"4

3'2

1"5

14,8

14'4

13'0

15'9

16'0

15'0

14'2

14'3

12'0

15'4

15'7

99

97

97

84

97

99

97

100

100

101

86

Values are expressed in fLmoles per 100 mg, of each sample on a moisture-free basis, Analytical methods are described in Methods section, * In the cases of CM-fractions, this stands for S-carboxymethylcysteine, t Values for 72 hours' hydrolysis.

Insect Biochem.

KAWASAKI AND OTHERS

dragonfly is covered by three morphologically distinct layers, which are produced by the follicle epithe1ium of the ovarium. The innermost homogeneous layer is the vitelline membrane, and this is in turn covered by two layers of chorion or egg-shell. The inner layer of chorion is thin and homogeneous, and the outer layer is thicker and filamentous. An electron micrograph of the laid egg (Fig. 2), taken in the present study, shows clearly the three layers. The thickness of the vitelline membrane and the inner and outer layers of chorion were estimated to be about 9, 0'3 and 3 fLm. respectively in S. infuscatum, and 7,0'25 and 2 fLm. respectively in S.frequens. When the crushed eggs were extracted with 0'01 M NaOH, ethanol and ether, the residues were found to contain all the three layers Table II.-COMPARISON OF THE AMINO-ACID COMPOSITION OF PROTEIN FRACTIONS OBTAINED FROM THE SCLEROPROTEIN FRACTIONS

S. infuscatum

GROUP OR RESIDUE

......

...... ......

......

I:i

I:i

I:i 0

.S...

.S...



U

U



..c


;l

C

U

..8-0 o· ... ......'"C p:::'"
.S ...0

56 '4 1"0 3·8 2'2 9'0 24'9 3'9 0

64'9 15'2 5'5 1·8 2'0 0 11"9 0·6

53'8 1"1 7'2 5'3 9'7 23'5 3'7 0

..::: :;8

..::: :;8

U

43. 6 0"8 16'7 14'3 13'4 21'3 3'7 0

1

Non-polar * Basic Total acidic t Amide Total hydroxyl t t Cystine Tyrosine Di- and tri tyrosine

S·frequens

1

..r:: U

C
.... ..c :::= S B
N.D.


I:i

..c ...... ...... I:i

u

.S... t)

.S...

..::: :;8


1

..::: :;8


1

..::: :;8

U

U

U

......I:i'" p:::'"
43'9 0"8 16'9 II'9 15'6 17'7 4·6 0

60·6 1'1 3.8 2'4 8"4 22'4 3"2 0

59'7 0·6 2'9 1"9 8'3 23'9 3'9 0

65'5 14'3 5'6 2'2 2·6 0 II'S 0'5

.~


t)

1


:0 ~ ..8-0 o· ...

Values are expressed in numbers of residues per 100 amino-acid residues, including di- and trityrosyl residues. Hexosamines are not included in this Table. Data for the chorion and vitelline membrane of S. infuscatum, prepared as described in the text, are also shown for comparison. * Proline, glycine, alanine, valine, methionine, isoleucine, leucine, and phenylalanine. t Aspartic acid, glutamic acid, and their amides. t Serine and threonine. N.D. = Not determined owing to the shortage of the sample. Values in this column were calculated without di- and trityrosine.

and the morphological properties of each layer seemed to be unchanged by the extractions. Thus, the scleroprotein fraction was assumed to contain the insoluble substances of both of the vitelline membrane and chorion. The amino-acid compositions of the sc1eroprotein fraction obtained from the two species of dragonfly are shown in Table I. The two fractions resembled each other c10sely in the chemical composition. Their phosphorus contents were negligible (less than 0'04 per cent). Dissolution and fractionation of the scleroprotein fraction revealed that it was almost exclusively made of proteins. The sum of the yields of the CM-fraction and insoluble residue was as much as 95 per cent of the scleroprotein fraction, even after the corrections

FIG, 2,-Electron micrograph of the egg envelopes of S,frequens, The laid egg which had been stored in a frozen state was fixed in 0'25 M glutaraldehyde/o' l M cacodylate buffer (pH 7'4)/0'2 M sucrose for 2 hours at 4° C, postfixed in 39 mM osmium tetraoxide-28 mM Veronal/acetate buffer (pH 7'4)-0'15 M sucrose for l hour at 40° C, dehydrated with ethanol, and embedded in Epon 812, Thin sections were stained with uranyl acetate and le ad citrate, OL and IL, are the outer and inner layers of chorion respectively; YT, vitelline membrane; OC, oocyte, x 5800,

CM~fraction fi

FIG, 3,-Free-boundary electrophoresis patterns (ascending) for CM-fractions 1 and II of S, infuscatum, Electrophoresis was carried out in a Tiselius-type apparatus, Hitachi HTD-I, at 3° C with buffers of ionic strength 0'2, The concentration of samples was about l ' l per cent, and aIl pictures shown were taken 60 minutes after starting electrophoresis, pH 8,6: Veronal/HCl/NaCI buffer, 3'10 V per cm, pH 6'1: phosphate buffer, 3'70 V per cm, pH 4'5: acetate/NaCI buffer, 3'54 V per cm,

CM-fraction 1

CM-fraction Il

FIG. 4.-Ultracentrifugal patterns for CM-fractions 1 and II of S. infuscatum. Samples (concentration, 1'1 per cent) were centrifuged at about 170,000g in a Hitachi UCA-IA analytical ultracentrifuge at zoo C. A bulb-type synthetic-boundary ceIl and 0'04 M phosphate/o'z M NaCI buffer (pH 8'0) as the solvent were used. The pictures shown were taken at bar-angle 65° about 80 minutes after reaching the set speed (SI,ZOO r.p.m.).

FIG. 5.-Acrylamide gel electrophoresis patterns for CM-fractions; 7'5 per cent gel (0'5 x 5 cm.) and 0'05 M Tris/o,z7 M glycine buffer (pH 8'3) were used. About 0'1 mg. of each protein was applied, and electrophoresis was carried out at room tempe rature with a constant current of Z mA. per gel for l' 5 hours. After electrophoresis, the gels were stained by toluidine blue without previous fixation. In the picture migration started at the upper ends of the gels and proceeded towards the lower ends. 1 and II, CM-fractions 1 and II of S. infuscatum respectively; If, lIa and lIb, CM-fractions l, lIa and lIb of S.frequens respectively.

PROTEINS IN DRAGONFLY EGG ENVELOPES

105

for the increased weight due to S-carboxymethylation. The ratio of the yields of CMfraction and insoluble residue was 24 : 76, and that of CM-fractions 1 and II was about 1 : 3 in both of the 2 species. Thus, when corrected for the chemical modification to obtain the CM-fraction, the scleroprotein fraction of S. infuscatum was assumed to consist of the three different groups of structural proteins in an approximate proportion of 1 : 3 : 14. In the case of S. frequens the CM-fraction II was further divided into two, and therefore its scleroprotein fraction was made up of four protein fractions, their ratio being about 1 : 1 : 2 : 14. PURITY OF THE PROTEIN FRACTIONS

The purity of the CM-fractions 1 and II of S. infuscatum was examined by freeboundary e1ectrophoresis, ultracentrifugation and acrylamide gel electrophoresis. Owing to the shortage of samples, the CM-fractions of S. frequens were tested only by gel e1ectrophoresis. CM-fractions 1 and II of S. infuscatum moved as a single boundary peak in free-boundary e1ectrophoresis (Fig. 3) and in ultracentrifugation (Fig. 4), but all of the protein Table III.-MoLEcuLAR PARAMETERS OF CM-FRACTIONS 1 AND II OF S. infuscatum PARAMETERS Sedimentation coefficient: s~o.w (Svedberg) Diffusion coefficient: D:u.w (10- 7cm. 2 sec.-1 ) Partial specific volume at zoo C: il (ml. per g.) Molecular weight * Frictional ratio *: f/fo Specific optical rotation: [am

CM-FRACTION 1

1'64 7'z 0'63

1'15

9'0 0'65

8,900

15,000

1'75 -80'5

CM-FRACTION II

± 1'5

1'91 0

- 84'5 ± l'5°

g. per 100 ml. (1'03 g. per 100 ml. of water, pH 5'6) of water, pH 5'6)

(0'75

Electrophoretic mobility t pH 8·6 6'1

(10- 5

cm. 2 V. -1 sec. -1)

4'5

* Calculated from s~o,w Dzoo,w and v.

t

Values calculated from the descending-boundary patterns of free-boundary electrophoresis were extrapolated to zero concentration of the samples. Buffers used were as in the legend to Fig. 3.

fractions showed heterogeneity in the gel electrophoresis (Fig. 5): 2 or 3 minor bands were detectable in aU samples. The major band of CM-fractions lIa and lIb was broad, although this was partly due to diffusion during the staining pro cess and the second major . band of CM-fraction II was a distinct one. These results may indicate that although each protein fraction consisted of proteins of similar general properties, the proteins were still heterogeneous mainly in molecular size or shape. We were unable to find a suitable solvent to dissolve the insoluble residue without cleaving peptide bonds, and its pu rity remains to be examined. 1 AND II OF S. infuscatum Physicochemical data obtained for CM-fractions 1 and II are shown in Table III. Because of the heterogeneity observed in the gel electrophoresis, the data represent only MOLECULAR PARAMETERS OF CM-FRACTIONS

106

KAWASAKI AND OTHERS

Inseet Bioehem.

the average values for the two protein fractions. Both of them were fibrous proteins of small molecular weight, but the average molecular weight of CM-fraction n was significantly higher than that of CM-fraction 1. CHEMICAL COMPOSITION OF THE PROTEIN FRACTIONS

The amino compositions of the protein fractions are listed in Tables 1 and n. In the case of S. infuseatum materials, the major protein fraction, the insoluble residue, contained large amounts of glycine, alanine, proline, and tyrosine and considerable amounts of lysine and histidine. It was remarkable that it contained di- and trityrosine but did not contain cystine at all. In contrast, the content of S-carboxymethylcysteine in the two soluble fractions, CM-fractions 1 and n, was very high, and the two contained only a small amount of lysine and lacked histidine and di- and trityrosine. AIso, their contents of alanine, proline and tyrosine were significantly lower than those of the insoluble residue, and there were additional differences in the relative amounts of serine, threonine, leucine, isoleucine and methionine. On the other hand, CM-fractions 1 and n differed from each other markedly in the contents of amide, glycine and alanine, although the relative amounts of S-carboxymethylcysteine, basic amino-acids and aromatic ones were similar. Thus, each of the three protein fractions had a quite different pattern of amino-acid composition. The general pattern of amino-acid composition for S. frequens fractions showed a close resemblance to that described above for the S. infuseatum fractions. CM-fractions IIa and IIb resembled each other and CM-fraction II of S. infuseatum more closely than CM-fraction 1. The important difference between IIa and IIb seemed to be different content of the total nitrogen and galactosamine, although there were considerable variations in the relative amounts of some of the amino-acids. CM-fractions were found to contain neutral sugars in addition to hexosamines, whereas none of neutral sugar was detectable in the insoluble residues. CM-fraction lof S. infuscatum contained, per 100 mg. of its weight, 6 fLmoles of galactose and 4 fLmoles of mannose, and CM-fraction II 15 fLmoles of galactose and a methylpentose (5 fLmoles as L-fucose). CM-fraction 1 of S. frequens contained only a small amount of hexose(s) (less than 4 fLmoles as galactose per 100 mg. sample), and we were not able to identify the hexose(s) in the present study. Neither pentose nor methylpentose was detectable in this fraction. In common with CM-fractions IIa and IIb, galactose, a pentose and a methylpentose were detected, but their contents differed remarkably: the contents of the hexose, pentose and methylpentose in 100 mg. of IIa were 8, 3 (as xylose) and 6 fLmoles (as L-fucose) respectively, but those in IIb were 25, 5 and 24 fLmoles respectively. ORIGIN OF THE PROTEIN FRACTIONS

The eggs which had been stored in a frozen state were placed in water. As the oocyte swelled under the vitelline membrane, the chorion split up and separated from the vitelline membrane. The chorions and the oocytes covered with the vitelline membrane could be collected separately under a binocular microscope. Collected chorions were washed with water, ethanol and ether before drying. The eggs without chorion were crushed, and the vitelline membranes were washed and dried as for the chorion. The chorion thus prepared was found to be readily soluble in the thiol-urea solvent used to fractionate the scleroprotein fraction, but the vitelline membrane was completely insoluble. Patterns of their amino-acid composition are shown in Table II. The vitelline

1974,4

PROTEINS IN DRAGONFLy EGG ENVELOPES

107

membrane did not contain cystine at aU, and the compositional pattern of other aminoacids was also similar to that of the insoluble residue. On the other hand, the amino-acid composition of the chorion resembled closely that of the combined CM-fractions 1 and II. Consequently, most of the insoluble residue, if not aU, must have originated from the vitelline membrane and the CM-fractions from the chorion. COMPARISON OF THE INSOLUBLE RESIDUES OBTAINED FROM THE YELLOW EGGS AND FROM THE DARK ONES

The initial difference in colour of the eggs was reflected only in the colour of the insoluble residues, and the coloured material, either yeUow or dark brown, was not extractable by various organic solvents teste d, such as acetone, benzene, ethanol, ether, methanoljchloroform (1 : 2, by volume), petrolium ether and phenol. The insoluble residue obtained from the dark eggs was more resistant against hydrolysis by acid, alkali or proteolytic enzyme than that from the yellow eggs. When the yeUow insoluble residue was heated in 1 M HCI or NaOH at 100 C, most of the material went into solution in about 1 hour, but most of the dark residue remained insoluble under the same conditions, and heating for an additional hour was necessary to dissolve most of it. Similarly, when a suspension of the yeUow insoluble residue in Tris buffer (pH 7) containing CaCl 2 to 10 mM was incubated with pro nase (sample : enzyme ratio, 100 : 1 by weight) at 37 0 C under toluene for 48 hours, 86 per cent of the material was converted to a soluble form, but under the same conditions the dark residue was practicaUy intact. As to the chemical composition, an interesting difference was observed between the two insoluble residues. Table 1 shows that the contents of tyrosine, lysine and histidine of the dark residue were significantly lower than those of the yellow, whereas the contents of other amino-acids, including di- and trityrosine, were similar in both. There was practicaUy no difference in the total nitrogen values of the two, but the recovery of nitrogen as amino-acids and amide was distinctly lower in the dark residue than in the yellow (Table 1). The acid hydrolysates of the two insoluble residues did not show any detectable unusual peak on the chart of the amino-acid analyser. However, when a hydrolysate of the dark one was applied to the analyser, a dark brown pigment remained at the top of the column and was not eluted with buffers. This phenomenon was also observed on the columns of DEAE-cellulose and cellulose phosphate. Similarly, most of the coloured material in the hydrolysate of the dark residue remained at the place of application wh en subjected to paper chromatography. A possibility that the yeUow insoluble residue might have contained sorne tyrosine, lysine and histidine as free amino-acids was improbable, because of the various extraction procedures used to prepare it. Further, 10 per cent trichloroacetic acid at 100 0 C did not extract an appreciable amount of any amino-acid from it during an extraction period of 30 minutes. These results suggested that the darkening process involved at least sorne of tyrosyl, lysyl and histidyl residues of the protein molecules in the formation of coloured chemical structures which stabilized the chemical organization of the vitelline membrane as intermolecular cross-links, and that the constituents of the structures were no longer released as free amino-acids by the hydrolysis with 6 M HCI at 100 0 C but probably aggregated together to form the dark brown pigment, which was strongly adsorbed by ion-exchangers and cellulose. 0

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OBSERVATIONS ON THE DARKENING OF EGGS

Sorne observations were made on the change of colour with the eggs collected in water. When the eggs were air-dried on filter-paper, the yellow colour began to change to brown after few hours and became dark brown after 24 hours. This pro cess, however, did not occur when the dried eggs were kept in vaeuo, in nitrogen gas or in air over a pyrogallol solution. Heating the eggs in water at 1000 C for 30 seconds or immersing themina 0·1 M solution ofKCN or NaN 3 for 10 minutes stopped the process completely. A1so, when crushed eggs were extracted with water, the residue failed to change colour. The darkening proceeded slowly in eggs under physiological conditions: in water and without shaking it took about a week for the completion of the process, but frequent shaking of the vessel or increasing the surface area of the suspension shortened the process considerably. On the other hand, when the chorion was removed from the egg manually or by dipping the egg in a thiol-urea solvent for few minutes, the process required only several hours. The eggs which were collected in water and kept frozen for 3 years did change to dark brown on exposure to air, but the eggs which had been treated by the freezingthawing procedure more than twice no longer changed colour, even if the eggs were newly laid. These observations may indicate the darkening process to be an enzymatic reaction requiring oxygen, and besides the nature of the enzyme, the access of oxygen to the vitelline membrane through the chorion is one of the important rate-limiting factors. in vitro BY TYROSINASE The observations described above suggested the possible participation of tyrosinase in the darkening process, which led us to experiment with the insoluble residue of S. infuseatum and mushroom tyrosinase (Miles-Seravac, 660 units per mg.). A 2 per cent suspension of the insoluble residue in 0'05 M phosphate buffer (pH 7.0) was added with the tyrosinase (sample : enzyme ratio, 20 : 1 by weight) and shaken at 300 C. Every 10 hours the insoluble residue was collected by centrifugation and was further shaken with renewed buffer and enzyme. The yellow colour of the sam pIe began to change to brown after about 10 hours and darkened slowly in the following 2 days. After 48 hours of incubation the colour was dark brown, although it was far less extensive than that of the insoluble residue obtained from the dark eggs. The sample was then washed with chilled 0·01 M NaOH, water, ethanol and ether, and dried. A suspension of the insoluble residue without added tyrosinase was shaken simultaneously as a control, and its colour was found to be unchanged. The amino-acid composition of the darkened insoluble residue showed that the contents of tyrosine, lysine and histidine were less by II, 7 and 4 fLmoles per 100 mg. of the sample respectively than those of the control sample, without any appreciable change in the contents of other amino-acids. Prolonged shaking of the sam pIe with frequent renewal of larger amounts of enzyme failed to produce more remarkable changes in the colour and in the contents of the three amino-acids. Thus, the decrease of tyrosine content in vitro was only about one-fifth of that observed in vivo (see Table 1), but the darkened sample became markedly resistant against digestion by pronase. Only 55 per cent of the sample went into solution by the action of pronase under the same condition as those described above, in which 86 per cent of the yellow insoluble residue was converted to a soluble form. Consequently, although the in vitro darkening process was limited considerably by DARKENING OF THE INSOLUBLE RESIDUE

PROTEINS IN DRAGONFLy EGG ENVELOPES

factors unknown to us at present, the mushroom tyrosinase did act on the insoluble residue, producing the changes in the colour, chemical composition and resistance against hydrolysis similar to the differences found between the insoluble residues obtained from the yellow eggs and from the dark ones. DISCUSSION

The major constituents of egg envelopes, the chorion and vitelline membrane, of the dragonfties studied were found to be structural proteins, as in the case of the chorions of silkworms (Kawasaki et al., 1971a, 1972) and of the oriental garden cricket (Kawasaki et al., 1971b). As with the major proteins in the chorions we have so far studied, the proteins in the dragonfty chorion were stabilized by the disulphide bonds of cystinyl residues. They were dissolved in a thiol-urea solvent and then modified to soluble S-carboxymethyl derivatives. Fractionation on a DEAE-cellulose column of the soluble derivatives of S. infuscatum gave two different protein fractions, CM-fractions 1 and II. They differed from each other particularly in the contents of amide, glycine, alanine and sugars and in the molecular weight, but both of them were fibrous proteins of small molecular weight and contained a large amount of S-carboxymethylcysteine. They did not contain phosphoserine, and thus their general properties resembled more the major proteins of silkworm chorions than that of the cricket chorion. CM-fraction II of S. frequens was obtained as two subfractions, lIa and lIb, which may be mainly due to the different content of sugars in these subfractions. Both CM-fraction 1 and II of S. infuscatum behaved as an essentially homogeneous protein in the ultracentrifugation and freeboundary electrophoresis, but the acrylamide gel electrophoresis indicated their heterogeneity. In the present study we did not attempt further purification of the samples, but it may become necessary for more detailed experiments. The origin of CM-fractions with respect to the two layers of chorion, and the relationship between CM-fractions 1 and II or between lIa and lIb are unknown at present. The vitelline membrane of the present material was far thicker than the chorion, in contrast to the eggs of silkworms and crickets. The proteins of the vitelline membrane, the insoluble residue, did not resemble in chemical composition any one of the thiol-ureainsoluble fractions obtained from the chorions of silkworms and crickets. The insoluble residue did not contain cystine at all, but it contained another type of intermolecular cross-link in the form of di- and trityrosyl residues. These cross-links have been found in two other insect structural proteins, resilin (Andersen, 1966) and Tussah silk fibroin (Raven, Earland & Little, 1971). Dityrosine has been found also in elastin from chick embryo aorta (LaBella, Keeley, Vivian & Thornhill, 1967) and in an alkali-soluble protein from bovine ligamentum nuchae (Keeley & LaBella, 1972), and both di- and trityrosine have been found in wool keratin (Raven et al., 1971), suggesting a possible wide distribution of these cross-links in structural proteins. In the present study we have also checked for their presence in sorne of the proteins so far prepared by us from chorions of silkworms and crickets. They were not detectable in any of the protein fractions that were soluble in thiol-urea solvents or in the thiourea-insoluble proteins (proteins III) of silkworms which were tested, Bombyx mori, Actias selene gnoma and Philosamia ricini. However, they were found in the thiol-urea-insoluble fraction (the insoluble residue) obtained from the cricket egg (Kawasaki et al., 1971 b): 0·6 fLmole of dityrosine and 0'2

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ILmole of trityrosine were present per 100 mg. of the sample. Consequently, this type of cross-link may also occur in other insect egg envelopes. The yellow colour of the newly laid eggs changed to dark brown after exposure to air. This darkening process was found to take place in the vitelline membrane, and it seemed to be a physiological one, since it occurred though slowly wh en the egg was placed in water. The chemical structure of the insoluble residue, which had been stabilized by di- and trityrosyl residues, was further reinforced by another type of cross-link during the darkening process. Results of the comparison between the chemical nature of the yellow insoluble residue and that of the dark one and findings on the in vitro darkening of the insoluble residue by mushroom tyrosinase indicated strongly that the darkening pro cess may be an unique form of quinone tanning, in which the tyrosyl residues of protein molecules, not free tyrosines, are oxidized to quinones to cross-link with free amino or imino groups of amino-acid residues. Although further study is necessary as to the detailed chemical nature of the cross-links, it is interesting to find evidence for the 'self-tanning' phenomenon in the vitelline membrane of the egg, since the' self-tanning' has been suggested to occur in insect cuticles by Hackman (1953) and Dennell & Malek (1955). The fact that the tyrosyl residue of proteins or peptides could be the substrate fortyrosinasehas beenshown byvarious authors (Sizer, 1948; Sizer& Wagley, 1951; Yasunobu, Peterson & Mason, 1959; Dabbous, 1966; Nishi & Tomura, 1969), and our present study showed that the enzyme acts also on the tyrosyl residue of an insoluble protein. In the hardening and darkening of insect cuticles and ootheca, small molecular substances have been regarded as important sclerotization agents. Pryor (1940) suggested that ortho di phenols are oxidized to quinone by a phenol oxidase in the cuticle and that the quinones then react spontaneously with available free amino or imino groups of protein molecules. Since then, N-acetyldopamine in the cuticle (Karlson & Sekeris, 1962) and protocatechuic acid (Pryor, Russell & Todd, 1946; Brunet & Kent, 1955) as well as 3,4-dihydroxybenzyl alcohol (Pau & Acheson, 1968) in cockroach ootheca have been found to be the important precursors of quinone. Andersen (1970, 1971) suggested, however, that quinones need not be intermediates in the hardening process of sorne insect cuticles, but that an orthodiphenol, such as N-acetyldopamine, is able to act as the sclerotization agent with its aliphatic side-chain functioning as the place of the cross-links. Therefore, considering these facts and evidence for' self-tanning', it seems that the darkening and hardening process in insects could be different according to where and when it takes place. A comparative study on the pro cess must be worthwhile chemically and physiologically, and the egg envelopes now seem to be an important group of materials for such study. ACKNOWLEDGEMENTS

We wish to thank Mr. M. Takashio, Iwate Medical College, for the electron microscope work. This work was supported in part by a Scientific Research Grant (768059) from the Ministry of Education of Japan. REFERENCES S. O. (1966), 'Covalent cross-links in a structural protein, resilin', Aeta physiol. seand., 66 (suppl. 263), 1-81. ANDERSEN, S. O. (1970), 'Isolation of arterenone (2-amino-3',4' -dihydroxyacetophenone) from hydrolysates of sclerotized insect cuticle', J. Inseet Physiol., 16, 1951-1959. ANDERSEN, S. O. (1971), 'Phenolic compounds isolated from insect hard cuticle and their relationship to the sclerotization process', Inseet Bioehem., l, 157-17°. ANDERSEN,

PROTEINS IN DRAGONFLy EGG ENVELOPES

III

BEAMS, H. W., & KESSEL, R. G. (1969), 'Synthesis and deposition of oocyte envelopes (vitelline membrane, chorion) and the uptake of yolk in the dragonfly (Odonata: Aeschnidae)', J. Cell Sei., 4, 241-264. BRUNET, P. C. J., & KENT, P. W. (1955), 'Observations on the mechanism of a tanning reaction in Periplaneta and Blatta', Proc. R. Soc. B, 144,259-274. DABBOUS, M. K. (1966), 'Inter- and intramolecular cross-linking in tyrosinase-treated tropocollagen',J. biol. Chem., 241, 53°7-5312. DAVIS, B. J. (1964), 'Disc electrophoresis. II. Method and application to human serum proteins', Ann. N. Y. Acad. Sei., 121,4°4-427. DENNELL, R., & MALEK, S. R. A. (1955), 'The cuticle of the cockroach Periplaneta americana. V. The chemical resistance of the impregnating material of the cuticle, and the "self-tanning" of its protein component', Proc. R. Soc. B, 144,545-556. GOODWIN, T. W., & MORTON, R. A. (1946), 'The spectrophotometric determination of tyrosine and tryptophan in proteins', Biochem. J., 40, 628-632. GROSS, A. J., & SIZER, 1. W. (1959), 'The oxidation of tyramine, tyrosine, and related compounds by peroxidase',J. biol. Chem., 234, 16II-1614. HACKMAN, R. H. (1953), 'Chemistry ofinsect cuticle. 3. Hardening and darkening of the cuticle', Biochem. J., 54, 371-377. KARLSON, P., & SEKERIS, C. E. (1962), 'N-Acetyldopamine as sclerotizing agent for the insect cuticle', Nature, Lond., 195, 183-184. KAWASAKI, H., SATO, H., & SUZUKI, M. (1971a), 'Structural proteins in the silkworm egg-shells', Insect Biochem., l, 13°-148. KAWASAKI, H., SATO, H., & SUZUKI, M. (1971b), 'Structural proteins in the egg-shell of the oriental garden cricket, Gryllus mitratus', Biochem.J., 125,495-5°5. KAWASAKI, H., SATO, H., & SUZUKI, M. (1972), 'Structural proteins in the egg-shell of silkworms, Bombyx mandarina and Antheraea mylitta', Insect Biochem., 2, 53-57. KEELEY, F. W., & LABELLA, F. S. (1972), 'Isolation of dityrosine from an alkali-soluble connective tissue protein', Biochim. biophys. Acta, 263, 52-59. LABELLA, F., KEELEY, F., VIVIAN, S., & THoRNHILL, D. (1967), 'Evidence for dityrosine in elastin', Biochem. biophys. Res. Commun., 26, 748-'753. MASAMUNE, H., & KAWASAKI, H. (1956), 'Chemical nature of KIK factors. 1. KIK factors in cancerous gastric juice', TohokuJ. exp. Med., 63, 369-382. MATSUBARA, H., & SASAKI, R. M. (1969, 'High recovery of tryptophan from acid hydrolysates of proteins', Biochem. biophys. Res. Commun., 35, 175-18I. MATSUZAKI, M. (1971), 'Electron microscopic studies on the oogenesis of dragonfly and cricket with special reference to the panoistic ovaries', Dev. Growth Differ., 13, 379-398. NISHI, H., & TOMuRA, R. (1969), 'Effects of tyrosinase on sericin',J. serie. Sci., Tokyo, 38, II7122. PAU, R. N., & ACHESON, R. M. (1968), 'The identification of 3-hydroxY-4-0-,B-D-glucosidobenzyl alcohol in the left colleterial gland of Blaberus discoidalis', Biochim. biophys. Acta, 158, 206-211. PRYOR, M. G. M. (1940), 'On the hardening of the cuticle of insects', Proc. R. Soc. B, 128, 393-4°7· PRYOR, M. G. M., RUSSELL, P. B., & TODD, A. R. (1946), 'Protocatechuic acid, the substance responsible for the hardening of the cockroach ootheca', Biochem. J., 40, 627-628. RAvEN, D. J., EARLAND, C., & LITTLE, M. (r971), 'Occurrence of dityrosine in Tussah silkfibroin and keratin', Biqchim. biophys. Acta, 25 1, 96-99. SIZER, 1. W. (1948), 'The inactivation of invertase by tyrosinase', Science, N. Y., 108, 335336. SIZER, 1. W., & WAGLEY, P. F. (1951), 'The action of tyrosinase on thrombin, fibrinogen, and fibrin',J. biol. Chem., 192,213-221. YASUNOBU, K. T., PETERSON, E. W., & MASON, H. S. (1959), 'The oxidation of tyrosine-containing peptides by tyrosinase', J. biol. Chem., 234, 3291-3295.

Key Word Index: Chorion, cross-links, dityrosine, dragonfly, egg envelopes, egg-shell, selftanning, structural proteins, Sympetrum frequens, Sympetrum infuscatum, trityrosine, tyrosinase, vitelline membrane.